In the field of cytology, individual cells can be differentiated on the basis of quantitative and qualitative characteristics, one of these characteristics being the cell's staining behavior. In techniques which evaluate staining behavior, the cell constituents to be measured, for example, DNA, RNA, and protein, are tagged with fluorescent dyes which fluoresce when illuminated, while the rest of the cell remains relatively dark at the wavelength of the fluorescence. The intensity of the fluorescent light and the amount or type of cell constituent are correlated so as to provide a basis for analysis of collected data. Consequently, it is important that the collected fluorescent signal corresponds to the amount of non-homogeneously or homogeneously distributed fluorescent material contained within the cell and not be dependent upon the cell's orientation and/or position in the illuminating radiation. Therefore, it readily may be seen that uniformity of illumination of the fluorescent material within a given cell is essential to obtaining accurate and reliable results.
As has recently become appreciated, illumination of cells with relatively narrow beams of illuminating radiation, such as laser light, creates "hot spots", i.e., regions of relatively large energy density as compared to neighboring regions within the cell. In other words, regions of non-uniform radiation or "hot spots" represent uneven illumination so that all parts of a cell are not exposed to the same amount of energy. These "hot spots" are due to optical effects at cell and organelle boundaries. This is particularily true of cells being irradiated by collimated radiation. Moreover, it is known in the art that converging beams, e.g., laser radiation, with a Gaussian intensity profile, become collimated in the focal region due to diffraction and therefore create the "hot spots" in the same manner. The problem with these "hot spots" is that if they coincide in location with the regions of fluorescent material within the cell, then that fluorescent material gives off a high intensity fluorescent signal relative to a low intensity fluorescent signal that the same fluorescent material would have produced if it had not been in the "hot spot". In short, if the "hot spot" is coincident with the fluorescent material, an inaccurate fluorescent reading is obtained.
The flow cytometers of the prior art, upon which the hereinafter described invention improves generally provide multi-parameter detection of stimulated fluorescent light and low-angle forward scatter light. A laser beam normally is used for fluorescence and scatter measurements, with the laser excitation beam being compressed in the direction of the fluid flow by beam shaping optics to achieve a desired thickness at the point of intersection with the particles. These particles are transported in suspension in a jet or flow stream through a measurement region in which the individually isolated particles are illuminated by the line focused or "slit-like" laser beam. The "slit-like" laser beam is used to minimize cell coincidence and to increase laser intensity. These systems use laminar sheath flow techniques for achieving a sequential flow of primarily single cells. Generally, two cylindrical lenses are utilized to create the "slit-like" laser beam, which comprises near-collimated light when impinging upon the particles. Consequently, it has been discovered that the "hot spot" problem previously described is inherent in this prior art design.
In the previously described prior art cytometers, less powerful laser beams lead to cost savings. Hence, it is desirable to provide a cytometer which efficiently uses the near-collimated laser beams commonly found in the prior art cytometers.
It readily can be seen that there is a need in the industry for an improved flow cytometer which more efficiently utilizes the laser beam and which has increased illumination from multiple directions without interfering with the fluorescence and forward scatter light collection.